Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

ABSTRACT

A cDNA encoding (E)-β-farnesene synthase from peppermint ( Mentha piperita ) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-β-farnesene synthase (SEQ ID NO:2), from peppermint ( Mentha piperita ). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-β-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-β-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-β-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-β-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-β-farnesene synthase may be used to obtain expression or enhanced expression of (E)-β-famesene synthase in plants in order to enhance the production of (E)-β-farnesene, or may be otherwise employed for the regulation or expression of (E)-β-farnesene synthase, or the production of its product.

RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.09/361,718, filed Jul. 27, 1999, now U.S. Pat. No. 6,258,602, which is acontinuation of prior application Ser. No. 09/166,460, filed Oct. 5,1998, now U.S. Pat. No. 6,008,043, priority from the filing date ofwhich is hereby claimed under 35 U.S.C. §120, and further claims thebenefit of provisional application No. 60/061,144, filed Oct. 6, 1997,the benefit of which is hereby claimed under 35 U.S.C. §119.

This invention was supported in part by the Department of Energy, GrantNo. DE-FG03-96ER20212; the National Institutes of Health, Grant No.GM-31354; and the Hatch Project, Grant No. 0268, from the AgriculturalResearch Center, Washington State University. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to nucleic acid sequences which code for(E)-β-farnesene synthases, such as the (E)-β-farnesene synthase fromMentha piperita, and to vectors containing the sequences, host cellscontaining the sequences and methods of producing recombinant(E)-β-farnesene synthases and their mutants.

BACKGROUND OF THE INVENTION

(E)-β-farnesene (FIG. 1) is an acyclic sesquiterpene olefin that occursin a wide range of both plant and animal taxa. Over 600 papers have beenpublished on the occurrence of this natural product and its deploymentas an important courier in chemical communication. The olefin is foundin the essential oil of hundreds of species of both gymnosperms, such asTorreya taxifolia (Florida torreya) (Shu. C. K., Lawrence, B. M. andCroom, E. M., Jr. (1995) J Essent. Oil Res. 7, 71-72) and Larixleptolepis (larch) (Nabeta, K., Ara, Y., Aoki, Y. and Miyake, M. (1990)J Nat. Prod. 53, 1241-1248), and angiosperms, such as Robiniapseudoacacia (black locust) (Kamden, D. P., Gruber, K., Barkman, L. andGage, D. A. (1994) J. Essent. Oil Res. 6, 199-200), Medicago sativa(alfalfa) (Kamm, J. A. and Buttery, R. G. (1983) Entomol. Exp. Appl. 33,129-134), Chamomilla recutita (chamomile) (Matos, P. J. A., Machiado, M.I. L., Alencar, J. W. and Craveiro, A. A. (1993) J. Essent. Oil Res. 5,337-339), Vitis vinifera (grapes) (Buchbauer, G., Jirovetz, L., Wasicky,M. and Nikiforov, A. (1994) J. Essent. Oil Res. 6, 311-314), Cannabissativa (hemp) (Lemberkovics, E., Veszki, P., Verzar-Petri, G. and Trka,A. (1981) Sci. Pharm. 49, 401-408), Zea mays (corn) (Turlings, T. C. J.,Tumlinson, J. H., Heath, R. R., Proveaux, A. T. and Doolittle, R. E.(1991) J. Chem. Ecol. 17, 2235-2251), Piper nigrum (black pepper),Daucus carota (carrot), and Mentha x piperita (peppermint) (Lawrence, B.M. (1972) Ann. Acad. Bras. Cienc. 44, (suppl.), 191-197).

While socially dominant male mice produce both α-farnesene and(E)-β-farnesene in their urine as pheromones (Novotny, M., Harvey, S.and Jemiolo, B. (1990) Experientia 46, 109-113), it is in the insectsand plants that the use of (E)-β-farnesene as a semiochemical is mostextensive. (E)-β-Farnesene is emitted by the Dufour's gland of andrenidbees (Fernandes, A., Duffield, R. M., Wheeler, J. W. and LaBerge, W. E.(1981) J. Chem. Ecol. 7, 453-460) and by several genera of ants (Ali, M.F., Morgan, E. D., Attygalle, A. B. and Billen, J. P. J. (1987) Z.Naturforsch. 42, 955-960; Jackson, B. D., Morgan, E. D. and Billen, J.P. J. (1990) Naturwiss. 77, 187-188; Ollet, D. G., Morgan, E. D.,Attygalle, A. B. and Billen, J. P. J. (1987) Z. Naturforsch. 42,141-146), where it serves both as a defensive allomone and as a trailpheromone. This sesquiterpene is synthesized de novo in the osmetrialglands of larval Papilio (Lepidoptera:Papilionidae) as an allomone(Honda, K. (1990) Insect Biochem. 20, 245-250), and it functions as afeeding stimulant to the sand fly Lutzomyia longipalpis(Diptera:Psychodidae), an important vector of the blood diseaseleislinaniasis (Tesh, R. B., Guzman, H. and Wilson, M. (1992) J Med.Entomol. 29, 226-231). Several species of predatory carabid beetles useE-β-farnesene as a prey-finding kairomone (Kielty, J. P.,Allen-Williams, L. J. Underwood, N. and Eastwood, E. A. (1996) J. InsectBehav. 9, 237-250). When released by corn, this olefin is also akairomonal oviposition stimulant to the European corn borer (Ostrinia)(Binder, B. F., Robbins, J. C. and Wilson. R. L. (1995) J. Chem. Ecol.21, 1315-1327). (E)-β-farnesene is the major component of pollen odor inLupinus and stimulates pollination behavior in bumblebees (Dobson, H. E.M., Groth, I. and Bergstroem, G. (1996) Am. J. Bot. 83, 877-885).Feeding by larval lepidopterans, such as Heliothis or Spodoptera(Noctuidae), increases the amount of (E)-β-farnesene released by corn;the volatile olefin is then detected as a synomone by the parasitic waspCotesia marginiventris (Hymenoptera:Braconidae) for locating thelepidopteran hosts (Turlings, T. C. J., Tumlinson, J. H., Heath, R. R.,Proveaux, A. T. and Doolittle, R. E. (1991) J. Chem. Ecol. 17,2235-2251). Circumstantial evidence also suggests the lepidopteraninduced production and emission of (E)-β-farnesene from corn serves as asynomone for Cotesia kariyai (Takabayashi, J., Takahashi, S., Dicke, M.and Posthumus, M. A. (1995) J. Chem. Ecol. 21, 273-287) and from cottonleaves as a synomone for C. marginiventris (Pare, P. W. and Tumlinson,J. H. (1997) Nature 385, 30-31; Loughrin, J. H., Manukian, A., Heath, R.R., Turlings, T. C. J. and Turnlinson, J. H. (1994) Proc. Natl. Acad.Sci. USA 91, 11836-11840).

Perhaps of greatest significance in plant-insect interactions is the useof E-β-farnesene by most aphid species as an alarm pheromone (Bowers, W.S., Nault, L. R., Webb, R. E. and Dutky, S. R. (1972) Science 177,1121-1122; Edwards, L. J., Siddall, J. B., Dunham, L. L., Uden, P. andKislow, C. J. (1973) Nature 241, 126-127). Aphids exposed to(E)-β-farnesene become agitated and disperse from their host plant(Wohlers, P. (1981) Z Angew. Entomol. 92, 329-336). Alate aphids areusually more sensitive than are apterae species and will often notcolonize a host displaying (E)-β-farnesene. Ants that defend aphids aresensitive to host-emitted (E)-βfarnesene and, when exposed, will displayaggressive behavior (Nault, L. R. and Montgomery, M. E. (1976) Science192, 1349-1351). (E)-β-farnesene also mimics the action of juvenilehormone III in some insects (Mauchamp, B. and Pickett. J. J. (1987)Agronomie 7, 523-529), may play a role in control of aphid morphologicaltypes, and is acutely toxic to aphids at a dose of 100 ng/aphid (vanOosten. A. M., Gut, J., Harrewijn, P. and Piron, P. G. M. (1990) ActaPhytopathol. Entomol. Hung. 25, 331-342). (E)-β-farnesene vapor is alsotoxic to whiteflies (Klijnstra, K. W., Corts, K. A. and van Oosten, A.M. (1992) Meded. Fac. Landbouwwet. 57, 485-491).

Efforts to control aphid behavior by topical application of(E)-β-farnesene to crops have met with little success, due to volatilityand rapid oxidative inactivation in air (Dawson, G. W., Griffiths, D.C., Pickett, J. A., Plumb, R. T., Woodcock. C. M. and Zhang, Z. N.(1988) Pest. Sci. 22, 17-30). Derivatives of (E)-β-farnesene withreduced volatility, or increased stability, have shown promise inreducing aphid-transmitted viruses, such as barley mosaic virus (Dawson,G. W., Griffiths, D. C., Pickett, J. A., Plumb, R. T., Woodcock, C. M.and Zhang Z. N. (1988) Pest. Sci 22, 17-30), potato virus Y (Gibson, R.W., Pickett, J. A., Dawson, G. W., Rice, A. D. and Stribley, M. F.(1984) Ann. Appl. Entomol. 104, 203-209), and beet mosaic virus (Gibson,R. W., Pickett, J. A., Dawson, G. W., Rice, A. D. and Stribley, M. F.(1984) Ann. Appl. Entomol. 104, 203-209). The wild potato Solanumberthaultii, which produces (E)-β-farnesene in type A trichomes, is morerepellent to the green peach aphid than are commercial varieties of S.tuberosum that produce lower levels of the olefin (Gibson, R. W. andPickett, J. A. (1983) Nature 302, 608-609; Ave, D. A., Gregory, P. andTingey, W. M. (1987) Entomol. Exp. App. 44, 131-138). In alfalfa,repellency to the blue alfalfa aphid and the pea aphid is correlatedwith the leaf content of (E)-β-farnesene, but not with the amount of theco-occurring sesquiterpene caryophyllene (Mostafavi, R., Henning, J. A.,Gardea-Torresday, J. and Ray, I. M. (1996) J. Chem. Ecol. 22,1629-1638).

For plants that produce (E)-β-farnesene, breeding for increasedproduction has met with some success (Mostafavi, R., Henning, J. A.,Gardea-Torresday, J. and Ray, I. M. (1996) J. Chem. Ecol 22, 1629-1638),but has been limited by genetic variation in these species.(E)-β-farnesene synthase has been purified from maritime pine (Pinuspinaster) and characterized (Salin, F., Pauly, G., Charon, J. andGleizes, M. (1995) J. Plant Phys. 146, 203-209), but the gene has notyet been isolated from any source. A cDNA clone for (E)-β-farnesenesynthase would, by transgenic manipulation, provide a valuable additionto the arsenal of natural compounds active in host plant resistance. Thesubstrate for (E)-β-farnesene synthase is farnesyl diphosphate, aubiquitous isoprenoid intermediate involved in cytoplasmic phytosterolbiosynthesis. Sesquiterpene synthases lack plastidial targetingsequences and are localized to the cytoplasm (Chappell, J. (1995) Annu.Rev. Plant Physiol. Plant Mol. Biol. 46, 521-547). Therefore, even inplants that do not normally produce sesquiterpenes, a recombinant(E)-β-farnesene synthase would be directed to the cytoplasm wheresubstrate is supplied by the mevalonate pathway and where production of(E)-β-farnesene should result.

SUMMARY OF THE INVENTION

In accordance with the foregoing, a cDNA encoding (E)-β-farnesenesynthase from peppermint (Mentha piperita) has been isolated andsequenced, and the corresponding amino acid sequence has been deduced.Accordingly, the present invention relates to isolated DNA sequenceswhich code for the expression of (E)-β-farnesene synthase, such as thesequence designated SEQ ID NO:1 which encodes an (E)-β-farnesenesynthase protein (SEQ ID NO:2) from peppermint (Mentha piperita).Additionally, the present invention relates to isolated, recombinant(E)-β-farnesene synthase proteins from peppermint (Mentha piperita). Inother aspects, the present invention is directed to replicablerecombinant cloning vehicles comprising a nucleic acid sequence, e.g., aDNA sequence which codes for an (E)-β-farnesene synthase, or for a basesequence sufficiently complementary to at least a portion of DNA or RNAencoding (E)-β-farnesene synthase to enable hybridization therewith(e.g., antisense RNA or fragments of DNA complementary to a portion ofDNA or RNA molecules encoding (E)-β-farnesene synthase which are usefulas polymerase chain reaction primers or as probes for (E)-β-farnesenesynthase or related genes). In yet other aspects of the invention,modified host cells are provided that have been transformed,transfected, infected and/or injected with a recombinant cloning vehicleand/or DNA sequence of the invention. Thus, the present inventionprovides for the recombinant expression of (E)-β-farnesene synthase, andthe inventive concepts may be used to facilitate the production,isolation and purification of significant quantities of recombinant(E)-β-farnesene synthase (or of its primary enzyme products) forsubsequent use, to obtain expression or enhanced expression of(E)-β-farnesene synthase in plants, microorganisms or animals, or may beotherwise employed in an environment where the regulation or expressionof (E)-β-farnesene synthase is desired for the production of thissynthase, or its enzyme product, or derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1. The sesquiterpene synthase substrate, farnesyl diphosphate, andsesquiterpene olefins found in peppermint essential oil.

FIG. 2. Radio-GC of the sesquiterpene olefins generated from[1-³H]farnesyl diphosphate by an enzyme preparation from peppermint oilgland secretory cells. The olefin fraction of steam-distilled peppermintoil was used as internal standard, and only the portion of thechromatogram containing the sesquiterpene olefins is shown.

FIG. 3A. GC-MS of the products generated from farnesyl diphosphate bythe recombinant (E)-β-farnesene synthase. Panel A: Total ionchromatogram. Numbered peaks are sesquiterpene olefins.

FIG. 3B. Mass spectrum and retention time of peak 1 designated in FIG.3A.

FIG. 3C. Mass spectrum and retention time of authentic (E)-β-farnesenefrom parley oil.

FIG. 3D. Mass spectrum and retention time of peak 6 designated in FIG.3A. The spectrum of this minor product is compromised by the low ionabundance and the corresponding prominence of background ions.

FIG. 3E. Mass spectrum and retention time of authentic δ-cadinene.

FIG. 4. Proposed mechanism for the formation of (E)-β-farnesene andδ-cadinene from famesyl diphosphate. OPP denotes the diphosphate moiety.Ionization of the enzyme-bound nerolidyl diphosphate intermediate andproton elimination can also produce (E)-β-farnesene.

FIG. 5. Monoterpene olefins generated from the alternate substrategeranyl diphosphate by recombinant (E)-β-farnesene synthase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the terms “amino acid” and “amino acids” refer to allnaturally occurring L-α-amino acids or their residues. The amino acidsare identified by either the single-letter or three-letter designations:

Asp D aspartic acid Ile I isoleucine Thr T threonine Leu L leucine Ser Sserine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine Pro Pproline His H histidine Gly G glycine Lys K lysine Ala A alanine Arg Rarginine Cys C cysteine Trp W tryptophan Val V valine Gln Q glutamineMet M methionine Asn N asparagine

As used herein, the term “nucleotide” means a monomeric unit of DNA orRNA containing a sugar moiety (pentose), a phosphate and a nitrogenousheterocyclic base. The base is linked to the sugar moiety via theglycosidic carbon (1′ carbon of pentose) and that combination of baseand sugar is called a nucleoside. The base characterizes the nucleotidewith the four bases of DNA being adeninie (“A”), guanine (“G”), cytosine(“C”) and thymine (“T”). Inosine (“I”) is a synthetic base that can beused to substitute for any of the four, naturally-occurring bases (A, C,G or T). The four RNA bases are A,G,C and uracil (“U”). The nucleotidesequences described herein comprise a linear array of nucleotidesconnected by phosphodiester bonds between the 3′ and 5′ carbons ofadjacent pentoses.

“Oligonucleotide” refers to short length single or double strandedsequences of deoxyribonucleotides linked via phosphodiester bonds. Theoligonucleotides are chemically synthesized by known methods andpurified, for example, on polyacrylamide gels.

The term “(E)-β-farnesene synthase” refers to an enzyme that is capableof converting farnesyl diphosphate to (E)-β-farnesene.

The term “essential oil plant,” or “essential oil plants,” refers to agroup of plant species that produce high levels of monoterpenoid and/orsesquiterpenoid and/or diterpenoid oils, and/or high levels ofmonoterpenoid and/or sesquiterpenoid and/or diterpenoid resins. Theforegoing oils and/or resins account for greater than about 0.005% ofthe fresh weight of an essential oil plant that produces them. Theessential oils and/or resins are more fully described, for example, inE. Guenther, The Essential Oils, Vols. I-VI, R. E. Krieger PublishingCo., Huntington N.Y., 1975, incorporated herein by reference. Theessential oil plants include, but are not limited to:

Lamiaceae, including, but not limited to, the following species: Ocimum(basil), Lavandula (Lavender), Origanum (oregano), Mentha (mint), Salvia(sage), Rosmecinus (rosemary), Thymus (thyme), Satureja and Monarda.

Umbelliferae, including, but not limited to, the following species:Carum (caraway), Anethum (dill), feniculum (fennel) and Daucus (carrot).

Asteraceae (Compositae), including, but not limited to, the followingspecies: Artemisia (tarragon, sage brush), Tanacetum (tansy).

Rutaceae (e.g., citrus plants); Rosaceae (eg., roses); Myrtaceae (e.g.,eucalyptus, Melaleuca); the Gramineae (e.g., Cymbopogon (citronella));Geranaceae (Geranium) and certain conifers including Abies (e.g.,Canadian balsam), Cedrus (cedar) and Thuja and Juniperus.

The range of essential oil plants is more fully set forth in E.Guenther, The Essential Oils, Vols. I-VI, R. E. Krieger Publishing Co.,Huntington N.Y, 1975, which is incorporated herein by reference.

The term “angiosperm” refers to a class of plants that produce seedsthat are enclosed in an ovary.

The term “gymnosperm” refers to a class of plants that produce seedsthat are not enclosed in an ovary.

Abbreviations used are: bp, base pairs; dpm, disintegrations per minute;DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FDP,farnesyl diphosphate; GC, gas chromatography; GDP, geranyl diphosphate;GGDP, geranylgeranyl diphosphate; I, identity; IPTG,isopropyl-β-D-thiogalactopyranoside; LB, Luria-Bertani; Mopso,3-(N-morpholino)-2-hydroxypropane-sulfonic acid; MS, mass spectrometry;PVPP, polyvinylpolypyrrolidone; S, similarity.

The term “percent identity” (% I) means the percentage of amino acids ornucleotides that occupy the same relative position when two amino acidsequences, or two nucleic acid sequences, are aligned side by side.

The term “percent similarity” (% S) is a statistical measure of thedegree of relatedness of two compared protein sequences. The percentsimilarity is calculated by a computer program that assigns a numericalvalue to each compared pair of amino acids based on chemical similarity(e.g., whether the compared amino acids are acidic, basic, hydrophobic,aromatic, etc.) and/or evolutionary distance as measured by the minimumnumber of base pair changes that would be required to convert a codonencoding one member of a pair of compared amino acids to a codonencoding the other member of the pair. Calculations are made after abest fit alignment of the two sequences has been made empirically byiterative comparison of all possible alignments. (Henikoff, S. andHenikoff, J. G., Proc. Nat'l Acad Sci USA 89: 10915-10919, 1992).

The abbreviation “SSC” refers to a buffer used in nucleic acidhybridization solutions. One liter of the 20× (twenty times concentrate)stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and88.2 g sodium citrate.

The terms “alteration”, “amino acid sequence alteration”, “variant” and“amino acid sequence variant” refer to (E)-β-farnesene synthasemolecules with some differences in their amino acid sequences ascompared to the corresponding, native, i.e., naturally-occurring,(E)-β-farnesene synthases. Ordinarily, the variants will possess atleast about 70% homology with the corresponding native (E)-β-farnesenesynthases, and preferably, they will be at least about 80% homologouswith the corresponding, native (E)-β-farnesene synthases. The amino acidsequence variants of the (E)-β-farnesene synthases falling within thisinvention possess substitutions. deletions, and/or insertions at certainpositions. Sequence variants of (E)-β-farnesene synthases may be used toattain desired enhanced or reduced enzymatic activity. modifiedregiochemistry or stereochemistry, or altered substrate utilization orproduct distribution.

Substitutional (E)-β-farnesene synthase variants are those that have atleast one amino acid residue in the native (E)-β-farnesene synthasesequence removed and a different amino acid inserted in its place at thesame position. The substitutions may be single, where only one aminoacid in the molecule has been substituted, or they may be multiple,where two or more amino acids have been substituted in the samemolecule. Substantial changes in the activity of the (E)-β-farnesenesynthase molecules of the present invention may be obtained bysubstituting an amino acid with a side chain that is significantlydifferent in charge and/or structure from that of the native amino acid.This type of substitution would be expected to affect the structure ofthe polypeptide backbone and/or the charge or hydrophobicity of themolecule in the area of the substitution.

Moderate changes in the activity of the (E)-β-farnesene synthasemolecules of the present invention would be expected by substituting anamino acid with a side chain that is similar in charge and/or structureto that of the native molecule. This type of substitution, referred toas a conservative substitution, would not be expected to substantiallyalter either the structure of the polypeptide backbone or the charge orhydrophobicity of the molecule in the area of the substitution.

Insertional (E)-β-farnesene synthase variants are those with one or moreamino acids inserted immediately adjacent to an amino acid at aparticular position in the native (E)-β-farnesene synthase molecule.Immediately adjacent to an amino acid means connected to either theα-carboxy or α-amino functional group of the amino acid. The insertionmay be one or more amino acids. Ordinarily, the insertion will consistof one or two conservative amino acids. Amino acids similar in chargeand/or structure to the amino acids adjacent to the site of insertionare defined as conservative. Alternatively, this invention includesinsertion of an amino acid with a charge and/or structure that issubstantially different from the amino acids adjacent to the site ofinsertion.

Deletional variants are those where one or more amino acids in thenative (E)-βfarnesene synthase molecules have been removed. Ordinarily,deletional variants will have one or two amino acids deleted in aparticular region of the (E)-β-farnesene synthase molecule.

The terms “biological activity”, “biologically active”, “activity” and“active” refer to the ability of the (E)-β-farnesene synthases of thepresent invention to catalyze the formation of (E)-β-farnesene fromfarnesyl diphosphate. (E)-β-farnesene synthase activity is measured inan enzyme activity assay, such as the assay described in Example 1herein. Amino acid sequence variants of the (E)-β-farnesene synthases ofthe present invention may have desirable altered biological activityincluding, for example, altered reaction kinetics, substrateutilization, product distribution or other characteristics such asregiochemistry and stereochemistry.

The terms “DNA sequence encoding”, “DNA encoding” and “nucleic acidencoding” refer to the order or sequence of deoxyribonucleotides along astrand of deoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the translated polypeptidechain. The DNA sequence thus codes for the amino acid sequence.

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit another piece of DNA (the insert DNA) such as, but not limited to, acDNA molecule. The vector is used to transport the insert DNA into asuitable host cell. The insert DNA may be derived from the host cell, ormay be derived from a different cell or organism. Once in the host cell,the vector can replicate independently of or coincidental with the hostchromosomal DNA, and several copies of the vector and its inserted DNAmay be generated. In addition, the vector contains the necessaryelements that permit translating the insert DNA into a polypeptide. Manymolecules of the polypeptide encoded by the insert DNA can thus berapidly synthesized.

The terms “transformed host cell,” “transformed” and “transformation”refer to the introduction of DNA into a cell. The cell is termed a “hostcell”, and it may be a prokaryotic or a eukaryotic cell. Typicalprokaryotic host cells include various strains of E. coli. Typicaleukaryotic host cells are plant cells, such as maize cells, yeast cells,insect cells or animal cells. The introduced DNA is usually in the formof a vector containing an inserted piece of DNA. The introduced DNAsequence may be from the same species as the host cell or from adifferent species from the host cell, or it may be a hybrid DNAsequence, containing some foreign DNA and some DNA derived from the hostspecies.

In accordance with the present invention, a cDNA (SEQ ID NO:1) encoding(E)-β-farnesene synthase (SEQ ID NO:2) from peppermint (Mentha piperita)was isolated and sequenced in the following manner. An enriched CDNAlibrary was constructed from peppermint secretory cell clustersconsisting of the eight glandular cells subtending the oil droplet.These cell clusters were harvested by leaf surface abrasion and the RNAcontained therein was isolated. mRNA was purified by oligo-dT cellulosechromatography, and 5 μg of mRNA was used to construct a λZAPII cDNAlibrary.

Plasmids were excised from the library en mass and used to transform E.coli strain XLOLR. Approximately 150 individual plasmid-bearing strainswere grown in 5 ml LB media overnight, and the corresponding plasmidswere purified before partial 5′-sequencing. Putative terpenoid synthasegenes were identified by sequence comparison using the BLAST program ofthe GCG Wisconsin Package ver. 8. Bluescript plasmids harboring uniquefull-length cDNA inserts with high similarity to known plant terpenoidsynthases were tested for functional expression following transformationinto E. coli XL1-Blue cells. A single extract, from the bacteriacontaining clone p43, including the cDNA insert set forth in SEQ IDNO:1, produced a sesquiterpene olefin from [1-³H]FDP, and this clone wasselected for further study.

A cell-free extract of E. coli XL-1 Blue cells harboring the plasmidp43, including the cDNA insert set forth in SEQ ID NO:1, was preparedand shown to be capable of catalyzing the divalent metal ion-dependentconversion of [1-³H]FDP to labeled sesquiterpene olefins. Controlreactions, employing extracts of XL1-Blue cells transformed withpBluescript lacking the insert, evidenced no detectable production ofsesquiterpene olefins from [1-³H]FDP, thereby demonstrating that a cDNAclone (SEQ ID NO:1) encoding (E)-β-farnesene synthase (SEQ ID NO:2) hadbeen acquired.

The recombinant (E)-β-farnesene synthase (SEQ ID NO:2) was inactive withthe C₂₀ substrate analog [1-³H]GGDP, but was able to catalyze thedivalent cation-dependent conversion of the C₁₀ analog [1-³H]GDP tomonoterpene olefins. Control reactions, employing extracts of XL1-Bluecells transformed with pBluescript lacking the insert, evidenced nodetectable production of monoterpene olefins from [1-³H]GDP, therebyconfirming that the monoterpene synthase activity expressed from thecDNA insert of p43 (SEQ ID NO:1) was a function of the (E)-β-farnesenesynthase (SEQ ID NO:2). This is the first report describing theutilization of GDP by a sesquiterpene synthase.

Complete sequencing of the (E)-β-farnesene synthase cDNA (SEQ ID NO:1)contained in p43 revealed an insert size of 1959 bp encoding an openreading frame of 550 amino acids with a deduced molecular weight of63,829. The deduced amino acid sequence of the (E)-β-farnesene synthase(SEQ ID NO:2) lacks a plastidial targeting peptide. Like all other knownterpenoid synthases, (E)-β-farnesene synthase is rich in tryptophan(1.8%) and arginine (5.5%) residues, and bears a DDXXD motif (SEQ IDNO:3) (residues 301-305 of SEQ ID NO:2) which is believed to coordinatethe divalent metal ion chelated to the substrate diphosphate group. Theenzyme has a deduced isoelectric point at pH 5.16.

The isolation of a cDNA (SEQ ID NO:1) encoding (E)-β-farnesene synthase(SEQ ID NO:2) permits the development of efficient expression systemsfor this functional enzyme; provides useful tools for examining thedevelopmental regulation of (E)-β-farnesene synthase; permitsinvestigation of the reaction mechanism(s) of this enzyme, and permitsthe isolation of other (E)-β-farnesene synthases. The isolation of an(E)-β-farnesene synthase cDNA (SEQ ID NO:1) also permits thetransformation of a wide range of organisms in order to enhance, enableor otherwise alter, the synthesis of (E)-β-farnesene.

Although the (E)-β-farnesene synthase protein set forth in SEQ ID NO:2lacks a plastidial targeting sequence, a targeting sequence from anotherprotein can be included in the (E)-β-farnesene synthase amino terminus.Transport sequences well known in the art (See, for example, thefollowing publications, the cited portions of which are incorporated byreference herein: von Heijne et al., Eur J. Biochem, 180:535-545, 1989;Stryer, Biochemistry, W. H. Freeman and Company, New York, N.Y., p. 769[1988]) may be employed to direct (E)-β-farnesene synthase to othercellular or extracellular locations.

In addition to the native (E)-β-farnesene synthase amino acid sequenceof SEQ ID NO:2, sequence variants produced by deletions, substitutions,mutations and/or insertions are intended to be within the scope of theinvention except insofar as limited by the prior art. The(E)-β-farnesene synthase amino acid sequence variants of this inventionmay be constructed by mutating the DNA sequences that encode thewild-type synthases, such as by using techniques commonly referred to assite-directed mutagenesis. Nucleic acid molecules encoding the(E)-β-farnesene synthases of the present invention can be mutated by avariety of PCR techniques well known to one of ordinary skill in theart. (See, for example, the following publications, the cited portionsof which are incorporated by reference herein: “PCR Strategies”, M. A.Innis, D. H. Gelfand and J. J. Sninsky, eds., 1995, Academic Press. SanDiego, Calif. (Chapter 14); “PCR Protocols: A Guide to Methods andApplications”, M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J.White. eds., Academic Press, NY (1990).

By way of non-limiting example, the two primer system utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into the (E)-β-farnesene synthasegenes of the present invention. Following denaturation of the targetplasmid in this system, two primers are simultaneously annealed to theplasmid; one of these primers contains the desired site-directedmutation, the other contains a mutation at another point in the plasmidresulting in elimination of a unique restriction site. Second strandsynthesis is then carried out, tightly linking these two mutations, andthe resulting plasmids are transformed into a mutS strain of E. coli.Plasmid DNA is isolated from the transformed bacteria, restricted withthe relevant restriction enzyme (thereby linearizing the unmutatedplasmids), and then retransformed into E. coli. This system allows forgeneration of mutations directly in an expression plasmid, without thenecessity of subcloning or generation of single-stranded phagemids. Thetight linkage of the two mutations and the subsequent linearization ofunmutated plasmids results in high mutation efficiency and allowsminimal screening. Following synthesis of the initial restriction siteprimer, this method requires the use of only one new primer type permutation site. Rather than prepare each positional mutant separately, aset of “designed degenerate” oligonucleotide primers can be synthesizedin order to introduce all of the desired mutations at a given sitesimultaneously. Transformants can be screened by sequencing the plasmidDNA through the mutagenized region to identify and sort mutant clones.Each mutant DNA can then be fully sequenced or restricted and analyzedby electrophoresis on Mutation Detection Enhancement gel (J. T. Baker)to confirm that no other alterations in the sequence have occurred (byband shift comparison to the unmutagenized control).

Again, by way of non-limiting example, the two primer system utilized inthe QuikChange™ Site-Directed Mutagenesis kit from Stratagene (LaJolla,Calif.). may be employed for introducing site-directed mutants into the(E)-β-farnesene synthase genes of the present invention. Double-strandedplasmid DNA, containing the insert bearing the target mutation site, isdenatured and mixed with two oligonucleotides complementary to each ofthe strands of the plasmid DNA at the target mutation site. The annealedoligonucleotide primers are extended using Pfu DNA polymerase, therebygenerating a mutated plasmid containing staggered nicks. Aftertemperature cycling, the unmutated, parental DNA template is digestedwith restriction enzyme DpnI which cleaves methylated or hemimethylatedDNA. but which does not cleave unmethylated DNA. The parental, templateDNA is almost always methylated or hemimethylated since most strains ofE.coli, from which the template DNA is obtained, contain the requiredmethylase activity. The remaining, annealed vector DNA incorporating thedesired mutation(s) is transformed into E. coli.

The mutated (E)-β-farnesene synthase gene can be cloned into a pET (orother) overexpression vector that can be employed to transform E. colisuch as strain E. coli BL21(DE3)pLysS, for high level production of themutant protein, and purification by standard protocols. Examples ofplasmid vectors and E. coli strains that can be used to express highlevels of the (E)-β-farnesene synthase proteins of the present inventionare set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual,2nd Edition (1989), Chapter 17. The method of FAB-MS mapping can beemployed to rapidly check the fidelity of mutant expression. Thistechnique provides for sequencing segments throughout the whole proteinand provides the necessary confidence in the sequence assignment. In amapping experiment of this type, protein is digested with a protease(the choice will depend on the specific region to be modified since thissegment is of prime interest and the remaining map should be identicalto the map of unmutagenized protein). The set of cleavage fragments isfractionated by microbore HPLC (reversed phase or ion exchange, againdepending on the specific region to be modified) to provide severalpeptides in each fraction. and the molecular weights of the peptides aredetermined by FAB-MS. The masses are then compared to the molecularweights of peptides expected from the digestion of the predictedsequence, and the correctness of the sequence quickly ascertained. Sincethe exemplary mutagenesis techniques set forth herein producesite-directed mutations, sequencing of the altered peptide should not benecessary if the mass spectrograph agrees with prediction. If necessaryto verify a changed residue, CAD-tandem MS/MS can be employed tosequence the peptides of the mixture in question, or the target peptidecan be purified for subtractive Edman degradation or carboxypeptidase Ydigestion depending on the location of the modification.

In the design of a particular site directed mutagenesis experiment, itis generally desirable to first make a non-conservative substitution(e.g., Ala for Cys, His or Glu) and determine if activity is greatlyimpaired as a consequence. The properties of the mutagenized protein arethen examined with particular attention to the kinetic parameters ofK_(m) and k_(cat) as sensitive indicators of altered function, fromwhich changes in binding and/or catalysis per se may be deduced bycomparison to the native enzyme. If the residue is by this meansdemonstrated to be important by activity impairment, or knockout, thenconservative substitutions can be made, such as Asp for Glu to alterside chain length, Ser for Cys, or Arg for His. For hydrophobicsegments, it is largely size that is usefully altered, althougharomatics can also be substituted for alkyl side chains. Changes in thenormal product distribution can indicate which step(s) of the reactionsequence have been altered by the mutation. Modification of thehydrophobic pocket can be employed to change binding conformations forsubstrates and result in altered regiochemistry and/or stereochemistry.

Other site directed mutagenesis techniques may also be employed with thenucleotide sequences of the invention. For example, restrictionendonuclease digestion of DNA followed by ligation may be used togenerate deletion variants of (E)-β-farnesene synthase, as described insection 15.3 of Sambrook et al. Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, New York, N.Y. [1989],incorporated herein by reference. A similar strategy may be used toconstruct insertion variants, as described in section 15.3 of Sambrooket al., supra.

Oligonucleotide-directed mutagenesis may also be employed for preparingsubstitution variants of this invention. It may also be used toconveniently prepare the deletion and insertion variants of thisinvention. This technique is well known in the art as described byAdelman et al. (DNA 2:183 [1983]); Sambrook et al., supra; “CurrentProtocols in Molecular Biology”, 1991, Wiley (NY), F. T. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. D. Seidman, J. A. Smith and K.Struhl, eds, incorporated herein by reference.

Generally, oligonucleotides of at least 25 nucleotides in length areused to insert, delete or substitute two or more nucleotides in the(E)-β-farnesene synthase molecule. An optimal oligonucleotide will have12 to 15 perfectly matched nucleotides on either side of the nucleotidescoding for the mutation. To mutagenize wild-type (E)-β-farnesenesynthase, the oligonucleotide is annealed to the single-stranded DNAtemplate molecule under suitable hybridization conditions. A DNApolymerizing enzyme, usually the Klenow fragment of E. coli DNApolymerase I, is then added. This enzyme uses the oligonucleotide as aprimer to complete the synthesis of the mutation-bearing strand of DNA.Thus, a heteroduplex molecule is formed such that one strand of DNAencodes the wild-type synthase inserted in the vector, and the secondstrand of DNA encodes the mutated form of the synthase inserted into thesame vector. This heteroduplex molecule is then transformed into asuitable host cell.

Mutants with more than one amino acid substituted may be generated inone of several ways. If the amino acids are located close together inthe polypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. An alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants:wild-type (E)-β-farnesene synthase DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

A gene encoding (E)-β-farnesene synthase may be incorporated into anyorganism (intact plant, animal, microbe, etc.), or cell culture derivedtherefrom that produces substrates that can be converted to(E)-β-farnesene. An (E)-β-farnesene synthase gene may be introduced intoany organism for a variety of purposes including, but not limited to:production of (E)-β-farnesene synthase, or its product (E)-β-farnesene;enhancement of the rate of production and/or the absolute amount of(E)-β-farnesene; enhancement of protection of plants against pests andpathogens, for example by producing (E)-β-farnesene to act as apollinator attractant synomone for predators and parasites of plantpests, or as an aphid alarm pheromone. While the nucleic acid moleculesof the present invention can be introduced into any organism. thenucleic acid molecules of the present invention will preferably beintroduced into a plant species.

Eukaryotic expression systems may be utilized for the production of(E)-β-farnesene synthase since they are capable of carrying out anyrequired posttranslational modifications and of directing the enzyme tothe proper cellular compartment. A representative eukaryotic expressionsystem for this purpose uses the recombinant baculovirus, Autographacalifornica nuclear polyhedrosis virus (AcNPV; M. D. Summers and G. E.Smith, A Manual of Methods for Baculovirus Vectors and Insect CellCulture Procedures [1986]; Luckow et al., Bio-technology, 6:47-55[1987]) for expression of the (E)-β-farnesene synthases of theinvention. Infection of insect cells (such as cells of the speciesSpodoptera frugiperda) with the recombinant baculoviruses allows for theproduction of large amounts of the (E)-β-farnesene synthase proteins. Inaddition, the baculovirus system has other important advantages for theproduction of recombinant (E)-β-farnesene synthase. For example,baculoviruses do not infect humans and can therefore be safely handledin large quantities. In the baculovirus system, a DNA construct isprepared including a DNA segment encoding (E)-β-farnesene synthase and avector. The vector may comprise the polyhedron gene promoter region of abaculovirus, the baculovirus flanking sequences necessary for propercross-over during recombination (the flanking sequences comprise about200-300 base pairs adjacent to the promoter sequence) and a bacterialorigin of replication which permits the construct to replicate inbacteria. The vector is constructed so that (i) the DNA segment isplaced adjacent (or operably linked or “downstream” or “under thecontrol of”) to the polyhedron gene promoter and (ii) thepromoter/(E)-β-farnesene synthase combination is flanked on both sidesby 200-300 base pairs of baculovirus DNA (the flanking sequences).

To produce the (E)-β-farnesene synthase DNA construct, a cDNA cloneencoding the full length (E)-β-farnesene synthase is obtained usingmethods such as those described herein. The DNA construct is contactedin a host cell with baculovirus DNA of an appropriate baculovirus (thatis, of the same species of baculovirus as the promoter encoded in theconstruct) under conditions such that recombination is effected. Theresulting recombinant baculoviruses encode the full (E)-β-farnesenesynthase. For example, an insect host cell can be cotransfected ortransfected separately with the DNA construct and a functionalbaculovirus. Resulting recombinant baculoviruses can then be isolatedand used to infect cells to effect production of the (E)-β-farnesenesynthase. Host insect cells include, for example, Spodoptera frugiperdacells, that are capable of producing a baculovirus-expressed(E)-β-farnesene synthase. Insect host cells infected with a recombinantbaculovirus of the present invention are then cultured under conditionsallowing expression of the baculovirus-encoded (E)-β-farnesene synthase.(E)-β-farnesene synthase thus produced is then extracted from the cellsusing methods known in the art.

Other eukaryotic microbes such as yeasts may also be used to practicethis invention. The baker's yeast Saccharomyces cerevisiae, is acommonly used yeast, although several other strains are available. Theplasmid YRp7 (Stinchcomb et al., Nature, 282:39 [1979]; Kingsman et al.,Gene 7:141 [1979]; Tschemper et al., Gene, 10:157 [1980]) is commonlyused as an expression vector in Saccharomyces. This plasmid contains thetrp1 gene that provides a selection marker for a mutant strain of yeastlacking the ability to grow in the absence of tryptophan, such asstrains ATCC No. 44,076 and PEP4-1 (Jones, Genetics, 85:12 [1977]). Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Yeast host cellsare generally transformed using the polyethylene glycol method, asdescribed by Hinnen (Proc. Natl. Acad. Sci. USA, 75:1929 [1978]).Additional yeast transformation protocols are set forth in Gietz et al.,N.A.R, 20(17):1425(1992); Reeves et al., FEMS, 99(2-3):193-197, (1992),both of which publications are incorporated herein by reference.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149 [1968]; Holland et al., Biochemistry, 17:4900 [1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In the construction ofsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3′ of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters that have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

Cell cultures derived from multicellular organisms, such as plants, maybe used as hosts to practice this invention. Transgenic plants can beobtained, for example, by transferring plasmids that encode(E)-β-farnesene synthase and a selectable marker gene, e.g., the kangene encoding resistance to kanamycin, into Agrobacterium tumifacienscontaining a helper Ti plasmid as described in Hoeckema et al., Nature,303:179-181 [1983] and culturing the Agrobacterium cells with leafslices, or other tissues or cells, of the plant to be transformed asdescribed by An et al., Plant Physiology, 81:301-305 [1986].Transformation of cultured plant host cells is normally accomplishedthrough Agrobacterium tumifaciens. Cultures of mammalian host cells andother host cells that do not have rigid cell membrane barriers areusually transformed using the calcium phosphate method as originallydescribed by Graham and Van der Eb (Virology, 52:546 [1978]) andmodified as described in sections 16.32-16.37 of Sambrook et al., supra.However, other methods for introducing DNA into cells such as Polybrene(Kawai and Nishizawa, Mol. Cell. Biol., 4:1172 [1984]), protoplastfusion (Schaffner, Proc. Natl. Acad. Sci USA, 77:2163 [1980]),electroporation (Neumann et al., EMBO J., 1:841 [1982]), and directmicroinjection into nuclei (Capecchi, Cell, 22:479 [1980]) may also beused. Additionally, animal transformation strategies are reviewed inMonastersky G. M. and Robl, J. M., Strategies in Transgenic AnimalScience, ASM Press, Washington, D.C., 1995, incorporated herein byreference. Transformed plant calli may be selected through theselectable marker by growing the cells on a medium containing, e.g.,kanamycin, and appropriate amounts of phytohormone such as naphthaleneacetic acid and benzytadenine for callus and shoot induction. The plantcells may then be regenerated and the resulting plants transferred tosoil using techniques well known to those skilled in the art.

In addition, a gene regulating (E)-β-farnesene synthase production canbe incorporated into the plant along with a necessary promoter which isinducible. In the practice of this embodiment of the invention, apromoter that only responds to a specific external or internal stimulusis fused to the target cDNA. Thus, the gene will not be transcribedexcept in response to the specific stimulus. As long as the gene is notbeing transcribed, its gene product and enzyme product are not produced.

An illustrative example of a responsive promoter system that can be usedin the practice of this invention is the glutathione-S-transferase (GST)system in maize. GSTs are a family of enzymes that can detoxify a numberof hydrophobic electrophilic compounds that often are used aspre-emergent herbicides (Weigand et al., Plant Molecular Biology,7:235-243 [1986]). Studies have shown that the GSTs are directlyinvolved in causing this enhanced herbicide tolerance. This action isprimarily mediated through a specific 1.1 kb mRNA transcription product.In short, maize has a naturally occurring quiescent gene already presentthat can respond to external stimuli and that can be induced to producea gene product. This gene has previously been identified and cloned.Thus, in one embodiment of this invention, the promoter is removed fromthe GST responsive gene and attached to an (E)-β-farnesene synthase genethat previously has had its native promoter removed. This engineeredgene is the combination of a promoter that responds to an externalchemical stimulus and a gene responsible for successful production of(E)-β-farnesene synthase.

In addition to the methods described above, several methods are known inthe art for transferring cloned DNA into a wide variety of plantspecies, including gymnosperms, angiosperms, monocots and dicots (see,e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRCPress, Boca Raton, Fla. [1993], incorporated by reference herein).Representative examples include electroporation-facilitated DNA uptakeby protoplasts in which an electrical pulse transiently permeabilizescell membranes, permitting the uptake of a variety of biologicalmolecules, including recombinant DNA (Rhodes et al., Science,240:204-207 [1988]); treatment of protoplasts with polyethylene glycol(Lyznik et al., Plant Molecular Biology, 13:151-161 [1989]); andbombardment of cells with DNA-laden microprojectiles which are propelledby explosive force or compressed gas to penetrate the cell wall (Kleinet al., Plant Physiol. 91:440-444 [1989] and Boynton et al., Science,240:1534-1538 [1988]). Transformation of Taxus species can be achieved,for example, by employing the methods set forth in Han et al, PlantScience, 95:187-196 (1994), incorporated by reference herein. A methodthat has been applied to Rye plants (Secale cereale) is to directlyinject plasmid DNA, including a selectable marker gene, into developingfloral tillers (de la Pena et al., Nature 325:274-276 (1987)). Further,plant viruses can be used as vectors to transfer genes to plant cells.Examples of plant viruses that can be used as vectors to transformplants include the Cauliflower Mosaic Virus (Brisson et al., Nature 310:511-514 (1984); Additionally, plant transformation strategies andtechniques are reviewed in Birch, R. G., Ann Rev Plant Phys Plant MolBiol, 48:297 (1997); Forester et al., Exp. Agric., 33:15-33 (1997). Theaforementioned publications disclosing plant transformation techniquesare incorporated herein by reference, and minor variations make thesetechnologies applicable to a broad range of plant species.

Each of these techniques has advantages and disadvantages. In each ofthe techniques, DNA from a plasmid is genetically engineered such thatit contains not only the gene of interest, but also selectable andscreenable marker genes. A selectable marker gene is used to select onlythose cells that have integrated copies of the plasmid (the constructionis such that the gene of interest and the selectable and screenablegenes are transferred as a unit). The screenable gene provides anothercheck for the successful culturing of only those cells carrying thegenes of interest. A commonly used selectable marker gene is neomycinphosphotransferase II (NPT II). This gene conveys resistance tokanamycin, a compound that can be added directly to the growth media onwhich the cells grow. Plant cells are normally susceptible to kanamycinand, as a result, die. The presence of the NPT II gene overcomes theeffects of the kanamycin and each cell with this gene remains viable.Another selectable marker gene which can be employed in the practice ofthis invention is the gene which confers resistance to the herbicideglufosinate (Basta). A screenable gene commonly used is theβ-glucuronidase gene (GUS). The presence of this gene is characterizedusing a histochemical reaction in which a sample of putativelytransformed cells is treated with a GUS assay solution. After anappropriate incubation, the cells containing the GUS gene turn blue.

The plasmid containing one or more of these genes is introduced intoeither plant protoplasts or callus cells by any of the previouslymentioned techniques. If the marker gene is a selectable gene, onlythose cells that have incorporated the DNA package survive underselection with the appropriate phytotoxic agent. Once the appropriatecells are identified and propagated, plants are regenerated. Progenyfrom the transformed plants must be tested to insure that the DNApackage has been successfully integrated into the plant genome.

Mammalian host cells may also be used in the practice of the invention.Examples of suitable mammalian cell lines include monkey kidney CVI linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line293S (Graham et al., J. Gen. Virol., 36:59 [1977]); baby hamster kidneycells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin,Proc. Natl. Acad. Sci USA 77:4216 [1980]); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243 [1980]); monkey kidney cells (CVI-76, ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); rat hepatomacells (HTC, MI.54, Baumann et al., J. Cell Biol., 85:1 [1980]); and TRIcells (Mather et al., Annals N.Y. Acad. Sci., 383:44 [1982]). Expressionvectors for these cells ordinarily include (if necessary) DNA sequencesfor an origin of replication, a promoter located in front of the gene tobe expressed, a ribosome binding site, an RNA splice site, apolyadenylation site, and a transcription terminator site.

Promoters used in mammalian expression vectors are often of viralorigin. These viral promoters are commonly derived from polyoma virus,Adenovirus 2, and most frequently Simian Virus 40 (SV40). The SV40 viruscontains two promoters that are termed the early and late promoters.These promoters are particularly useful because they are both easilyobtained from the virus as one DNA fragment that also contains the viralorigin of replication (Fiers et al., Nature, 273:113 [1978]). Smaller orlarger SV40 DNA fragments may also be used, provided they contain theapproximately 250-bp sequence extending from the HindIII site toward theBgII site located in the viral origin of replication.

Alternatively, promoters that are naturally associated with the foreigngene (homologous promoters) may be used provided that they arecompatible with the host cell line selected for transformation.

An origin of replication may be obtained from an exogenous source, suchas SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and insertedinto the cloning vector. Alternatively, the origin of replication may beprovided by the host cell chromosomal replication mechanism. If thevector containing the foreign gene is integrated into the host cellchromosome, the latter is often sufficient.

The use of a secondary DNA coding sequence can enhance production levelsof (E)-β-farnesene synthase in transformed cell lines. The secondarycoding sequence typically comprises the enzyme dihydrofolate reductase(DHFR). The wild-type form of DHFR is normally inhibited by the chemicalmethotrexate (MTX). The level of DHFR expression in a cell will varydepending on the amount of MTX added to the cultured host cells. Anadditional feature of DHFR that makes it particularly useful as asecondary sequence is that it can be used as a selection marker toidentify transformed cells. Two forms of DHFR are available for use assecondary sequences, wild-type DHFR and MTX-resistant DHFR. The type ofDHFR used in a particular host cell depends on whether the host cell isDHFR deficient (such that it either produces very low levels of DHFRendogenously, or it does not produce functional DHFR at all).DHFR-deficient cell lines such as the CHO cell line described by Urlauband Chasin, supra, are transformed with wild-type DHFR coding sequences.After transformation, these DHFR-deficient cell lines express functionalDHFR and are capable of growing in a culture medium lacking thenutrients hypoxanthine, glycine and thymidine. Nontransformed cells willnot survive in this medium.

The MTX-resistant form of DHFR can be used as a means of selecting fortransformed host cells in those host cells that endogenously producenormal amounts of functional DHFR that is MTX sensitive. The CHO-K1 cellline (ATCC No. CL 61) possesses these characteristics, and is thus auseful cell line for this purpose. The addition of MTX to the cellculture medium will permit only those cells transformed with the DNAencoding the MTX-resistant DHFR to grow. The nontransformed cells willbe unable to survive in this medium.

Prokaryotes may also be used as host cells for the initial cloning stepsof this invention. They are particularly useful for rapid production oflarge amounts of DNA, for production of single-stranded DNA templatesused for site-directed mutagenesis, for screening many mutantssimultaneously, and for DNA sequencing of the mutants generated.Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No.31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No.31,537), and E. coli B; however many other strains of E coli, such asHB101, JM101, NM522, NM538, NM539, and many other species and genera ofprokaryotes including bacilli such as Bacillus subtilis, otherenterobacteriaceae such as Salmonella typhimurium or Serralia marcesans,and various Pseudomonas species may all be used as hosts. Prokaryotichost cells or other host cells with rigid cell walls are preferablytransformed using the calcium chloride method as described in section1.82 of Sambrook et al., supra. Alternatively, electroporation may beused for transformation of these cells. Prokaryote transformationtechniques are set forth in Dower. W. J., in Genetic Engineering,Principles and Methods, 12:275-296, Plenum Publishing Corp., 1990;Hanahan et al., Meth. Enzymol., 204:63 (1991).

As a representative example, cDNA sequences encoding (E)-β-farnesenesynthase may be transferred to the (His)₆.Tag pET vector commerciallyavailable (from Novagen) for overexpression in E. coli as heterologoushost. This pET expression plasmid has several advantages in high levelheterologous expression systems. The desired cDNA insert is ligated inframe to plasmid vector sequences encoding six histidines followed by ahighly specific protease recognition site (thrombin) that are joined tothe amino terminus codon of the target protein. The histidine “block” ofthe expressed fusion protein promotes very tight binding to immobilizedmetal ions and permits rapid purification of the recombinant protein byimmobilized metal ion affinity chromatography. The histidine leadersequence is then cleaved at the specific proteolysis site by treatmentof the purified protein with thrombin, and the (E)-β-farnesene synthaseagain purified by immobilized metal ion affinity chromatography, thistime using a shallower imidazole gradient to elute the recombinantsynthases while leaving the histidine block still adsorbed. Thisoverexpression-purification system has high capacity, excellentresolving power and is fast, and the chance of a contaminating E. coliprotein exhibiting similar binding behavior (before and after thrombinproteolysis) is extremely small.

As will be apparent to those skilled in the art, any plasmid vectorscontaining replicon and control sequences that are derived from speciescompatible with the host cell may also be used in the practice of theinvention. The vector usually has a replication site, marker genes thatprovide phenotypic selection in transformed cells, one or morepromoters, and a polylinker region containing several restriction sitesfor insertion of foreign DNA. Plasmids typically used for transformationof E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and BluescriptM13, all of which are described in sections 1.12-1.20 of Sambrook etal., supra. However, many other suitable vectors are available as well.These vectors contain genes coding for ampicillin and/or tetracyclineresistance which enables cells transformed with these vectors to grow inthe presence of these antibiotics.

The promoters most commonly used in prokaryotic vectors include theβ-lactamase (penicillinase) and lactose promoter systems (Chang et al.Nature, 375:615 [1978]; Itakura et al., Science, 198:1056 [1977];Goeddel et al., Nature, 281:544 [1979]) and a tryptophan (trp) promotersystem (Goeddel et al., Nucl. Acids Res., 8:4057 [1980]; EPO Appl. Publ.No. 36,776), and the alkaline phosphatase systems. While these are themost commonly used, other microbial promoters have been utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally into plasmidvectors (see Siebenlist et al., Cell, 20:269 [1980]).

Many eukaryotic proteins normally secreted from the cell contain anendogenous secretion signal sequence as part of the amino acid sequence.Thus, proteins normally found in the cytoplasm can be targeted forsecretion by linking a signal sequence to the protein. This is readilyaccomplished by ligating DNA encoding a signal sequence to the 5′ end ofthe DNA encoding the protein and then expressing this fusion protein inan appropriate host cell. The DNA encoding the signal sequence may beobtained as a restriction fragment from any gene encoding a protein witha signal sequence. Thus, prokaryotic, yeast, and eukaryotic signalsequences may be used herein, depending on the type of host cellutilized to practice the invention. The DNA and amino acid sequenceencoding the signal sequence portion of several eukaryotic genesincluding, for example, human growth hormone, proinsulin, and proalbuminare known (see Stryer, Biochemistry W. H. Freeman and Company, New York,N.Y., p. 769 [1988]), and can be used as signal sequences in appropriateeukaryotic host cells. Yeast signal sequences, as for example acidphosphatase (Arima et al., Nuc. Acids Res., 11:1657 [1983]), α-factor,alkaline phosphatase and invertase may be used to direct secretion fromyeast host cells. Prokaryotic signal sequences from genes encoding, forexample, LamB or OmpF (Wong et al., Gene, 68:193 [1988]), MalE, PhoA, orbeta-lactamase, as well as other genes, may be used to target proteinsfrom prokaryotic cells into the culture medium.

Trafficking sequences from plants, animals and microbes can be employedin the practice of the invention to direct the (E)-β-farnesene synthaseproteins of the present invention to the cytoplasm, endoplasmicreticulum, mitochondria or other cellular components, or to target theprotein for export to the medium. These considerations apply to theoverexpression of (E)-β-farnesene synthase, and to direction ofexpression within cells or intact organisms to permit gene productfunction in any desired location.

The construction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes and the(E)-β-farnesene synthase DNA of interest are prepared using standardrecombinant DNA procedures. Isolated plasmids and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors, as is well known in the art (see, for example,Sambrook et al., supra).

As discussed above, (E)-β-farnesene synthase variants are preferablyproduced by means of mutation(s) that are generated using the method ofsite-specific mutagenesis. This method requires the synthesis and use ofspecific oligonucleotides that encode both the sequence of the desiredmutation and a sufficient number of adjacent nucleotides to allow theoligonucleotide to stably hybridize to the DNA template.

The foregoing may be more fully understood in connection with thefollowing representative examples, in which “Plasmids” are designated bya lower case p followed by an alphanumeric designation. The startingplasmids used in this invention are either commercially available,publicly available on an unrestricted basis, or can be constructed fromsuch available plasmids using published procedures. In addition, otherequivalent plasmids are known in the art and will be apparent to theordinary artisan.

“Digestion”, “cutting” or “cleaving” of DNA refers to catalytic cleavageof the DNA with an enzyme that acts only at particular locations in theDNA. These enzymes are called restriction endonucleases, and the sitealong the DNA sequence where each enzyme cleaves is called a restrictionsite. The restriction enzymes used in this invention are commerciallyavailable and are used according to the instructions supplied by themanufacturers. (See also sections 1.60-1.61 and sections 3.38-3.39 ofSambrook et al., supra.)

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the resulting DNA fragment on apolyacrylamide or an agarose gel by electrophoresis, identification ofthe fragment of interest by comparison of its mobility versus that ofmarker DNA fragments of known molecular weight, removal of the gelsection containing the desired fragment, and separation of the gel fromDNA. This procedure is known generally. For example, see Lawn et al.(Nucleic Acids Res., 9:6103-6114 [1982]), and Goeddel et al. (NucleicAcids Res., supra).

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1 Essential Oil Analysis and Cell-Free Assay

Plant Material and Reagents. Unless stated otherwise, the followingplant materials and reagents were used in the experiments reported inthis and succeeding Examples. Mentha x piperita L. cv. ‘Black Mitcham’was propagated from rhizomes as previously described (Gershenzon, J.,McCaskill, D., Rajaonarivony, J. I. M., Mihaliak, C., Karp, F. andCroteau, R. (1992) Anal. Biochem. 200, 130-138). The preparations of[1-³H]geranyl diphosphate (GDP) (250 Ci/mol), [1-³H]farnesyl diphosphate(FDP) (125 Ci/mol), and [1-³H]geranylgeranyl diphosphate (GGDP) (118Ci/mol) have been previously reported (Croteau, R., Alonso, W. R.,Koepp, A. E. and Johnson, M. A. (1994) Arch. Biochem. Biophys. 309,184-192; Dixit, V. M., Laskovics, F. M., Noall, W. I. and Poulter, C. D.(1981) J. Org. Chem. 46, 1967-1969; LaFever, R. E., StoferVogel, B. andCroteau, R. (1994) Arch. Biochem. Biophys. 313, 139-149). Terpenoidstandards were from our own collection or were prepared from plantmaterial purchased locally. α-Farnesene was a gift from Dr. J. Brown(Washington State University), δ-cadinene was a gift from Dr. M.Essenberg (Oklahoma State University), and commercially steam distilledpeppermint oil was a gift from I. P. Callison and Sons, Inc., Chehalis,Wash. All other biochemicals and reagents were purchased from SigmaChemical Co. or Aldrich Chemical Co., unless otherwise noted.

Sesquiterpene Analysis. Unless stated otherwise, the following procedurewas utilized to analyze sesquiterpene content and composition in theexperiments reported in this and succeeding Examples. Young, maturepeppermint leaves were harvested and hydrodistilled fromNH₄HCO₃-buffered water with simultaneous pentane extraction (Maarse, H.and Kepner, R. E. (1970) J. Agr. Chem. 18, 1095-1101). The organic phasewas passed through a column of MgSO₄-silica gel (MallinckrodtSilicAR-60) to provide the olefin fraction for GC-MS analysis. Authentic(E)-β-farnesene was prepared by pentane extraction (followed by silicagel fractionation) of macerated ginger (Zingiber officinale) root, blackpepper oleoresin (Piper nigrum), bergamot oil (Citrus bergarmot),parsley oil (Petroselinum crispum), or field-grown (Yakima Valley,Wash.) commercial peppermint oil (Lawrence. B. M. (1972) Ann. Acad.Bras. Cienc. 44, (suppl.), 191-197); all of these sources are reportedto contain (E)-β-farnesene.

Instrumental Analysis. The following instrumentation was utilized inthis Example and all succeeding Examples, unless stated otherwise.Radio-GC was performed on a Gow-Mac 550P instrument (He carrier 40ml/min, injector 220° C. detector 250° C. and 150 mA) attached to aPackard 894 gas proportional counter. The column (3.18 mm i.d. by 3.66 mstainless steel with 15% polyethylene glycol ester (AT1000 Alltech) onGas Chrom Q was programmed from 150° C. (5 min. hold) to 220° C. at 5°C./min. Thermal conductivity and radioactivity outputs were monitoredafter calibration with an external radiochemical standard, and ˜20,000dpm of tritiated product was injected with data analysis usingTurbochrome Navigator ver. 4.1 software (Perkin-Elmer). Liquidscintillation counting was performed in toluene:ethanol (70:30, v/v)containing 0.4% Omnifluor (DuPont NEN) using a Packard 460 CDspectrometer (³H efficiency ˜43%). GC-MS analysis employed aHewlett-Packard 6890-5972 system with a 5MS capillary column (0.25 mmi.d. by 30 m with 0.25 μm coating of 5% phenyl methyl siloxane).Injections were made cool on-column at 40° C. with oven programming from40° C. (50° C./min) to 50° C. (5 min hold), then 10° C./min to 250° C.,then 50° C./min to 300° C. Separations were made under a constant flowof 0.7 ml He/min. Mass spectral data were collected at 70 eV andanalyzed using Hewlett-Packard Chemstation software.

Cell-Free Assays. Peppermint oil gland secretory cells were isolatedfrom immature leaves as previously described (Gershenzon, J., McCaskill,D., Rajaonarivony, J. I. M., Mihaliak, C., Karp, F. and Croteau, R.(1992) Anal. Biochem. 200, 130-138, incorporated herein by reference)and sonically disrupted (Braun-Sonic 2000 microprobe at maximum powerfor three 30-second bursts with 30-second chilling period at 0-4° C.between bursts) into assay buffer consisting of 25 mM Mopso (pH 7.0), 10mM sodium ascorbate, 25 mM KCl, 10 mM DTT and 10% glycerol, andsupplemented with 0.5% (w/v) PVPP and 1% (w/v) Amberlite XAD-4polystyrene resin. The sonicate was centrifuged at 3700×g for 15minutes, and an aliquot of the supernatant was then placed in a 10 mlscrew-capped glass test tube containing divalent metal ions (10 mM MgCl₂and 1 mM MnCl₂) and substrate (7.3 μM [1-³H]FDP). The aqueous layer wasoverlaid with 1 ml pentane and the seated tube was incubated at 30° C.for two hours. The pentane overlay was then collected and the aqueouslayer was extracted twice (1 ml) with pentane. The combined pentaneextracts were passed through an anhydrous MgSO₄-silica gel column toobtain the labeled hydrocarbon fraction for GC-MS analysis, or forradio-GC analysis using peppermint oil as an internal standard.

Essential Oil Analysis. To assess the probable abundance of(E)-β-farnesene synthase in peppermint gland secretory cells, theexclusive site of essential oil biosynthesis (Gershenzon, J., McCaskill,D., Rajaonarivony, J. I. M., Mihaliak, C., Karp, F. and Croteau, R.(1992) Anal. Biochem. 200, 130-138) the sesquiterpene olefin fraction offield-distilled peppermint oil was analyzed by GC-MS and shown tocontain β-caryophyllene (39%), γ-cadinene (33%), β-bourbonene (11%),(E)-β-farnesene (2.9%), δ-cadinene (2.0%), germacrene D (1.3%), copaene(1.3%) and α-humulene (1.2%) (FIG. 1), as well as several other minorcomponents (<1% each). GC-MS analysis of the oil distilled fromgreenhouse material revealed a similar composition, except that theamount of γ-cadinene was higher (53%), β-bourbonene was conspicuouslyabsent, and the (E)-β-farnesene content was 3.4%. Although(E)-β-frnesene was not one of the more prominent sesquiterpenes ofpeppermint, the abundance was sufficient to suggest that cloning of thecorresponding synthase by random sequencing of an enriched, oil glandcDNA library might be possible.

Cell-free extracts. To gain a preliminary assessment of the targetactivity, cell-free extracts of peppermint oil gland secretory cells(Gershenzon, J., McCaskill, D., Rajaonarivony, J. I. M., Mihaliak, C.,Karp, F. and Croteau, R. (1992) Anal. Biochem. 200, 130-138), wereassayed for the divalent metal ion-dependent conversion of[1-³H]farnesyl diphosphate to sesquiterpene olefins (Cane, D. E. (1990)Chem. Rev. 90, 1089-1103). Radio-GC analysis of the derived biosyntheticproducts (FIG. 2) revealed the presence of two major componentsidentified as caryophyllene and γ-cadinene. However, the separation ofthe labeled olefins was insufficient to resolve (E)-β-farnesene fromcaryophyllene, or δ-cadinene-from γ-cadinene. Both of these minorcomponents appear at the trailing edges of the major peaks but are,nevertheless, coincident with the authentic standards, indicating thecorresponding biosynthetic capability. No β-bourbonene was synthesizedfrom FDP by this system.

EXAMPLE 2 Cloning and Expression in E. coli of a cDNA Encoding(E)-β-Farnesene Synthase (SEQ ID NO:1)

Library Construction and Clone Identification. Initial cloning offull-length terpenoid biosynthetic genes from the peppermint oil glandcDNA library was successful and established a very high degree ofenrichment for these target sequences. For example, the monoterpenecyclase, limonene synthase (Colby, S. M., Alonso, W. R., Katahira, E.J., McGarvey, D. J. and Croteau, R. (1993) J. Biol. Chem. 268,23016-23024), represents approximately 4% of the library. This fact,plus the availability of automated sequencing capability, led to thepossibility of randomly sequencing the library in search of cDNA speciesencoding other terpenoid synthases, including the (E)-β-farnesenesynthase which was shown to be operational in this plant by bothsesquiterpene analysis and cell-free assay.

An enriched cDNA library was constructed from peppermint secretory cellclusters consisting of the eight glandular cells subtending the oildroplet. These cell clusters were harvested by a leaf surface abrasiontechnique (Gershenzon, J., McCaskill, D., Rajaonarivony, J. I. M.,Mihaliak, C., Karp, F. and Croteau, R. (1992) Anal. Biochem. 200,130-138), and the RNA contained therein was isolated using the protocolof Logemann et al. (Logemann, J., Schell, J. and Willmitzer, L. (1987)Anal. Biochem. 163, 16-20). mRNA was purified by oligo-dT cellulosechromatography (Pharmacia), and 5 μg of mRNA was used to construct aλZAPII cDNA library according to the manufacturer's instructions(Stratagene).

Plasmids were excised from the library en mass and used to transform E.coli strain XLOLR as per the manufacturer's instructions (Stratagene).Approximately 150 individual plasmid-bearing strains were grown in 5 mlLB media overnight, and the corresponding plasmids were purified using aQiawell 8 Ultraplasmid Kit (Qiagen) before partial 5′-sequencing by theDye-Deoxy™ method using an ABI Sequenator at the Laboratory forBiotechnology and Bioanalysis at Washington State University. Putativeterpenoid synthase genes were identified by sequence comparison usingthe BLAST program of the GCG Wisconsin Package ver. 8. Bluescriptplasmids harboring unique full-length cDNA inserts with high similarityto known plant terpenoid synthases were tested for functional expressionfollowing transformation into E. coli XL1-Blue cells. A single extract,from the bacteria containing clone p43, including the cDNA insertsequence set forth in SEQ ID NO:1, produced a sesquiterpene olefin from[1-³H]FDP, and this clone was selected for further study.

Bacterial Expression and Characterization of (E)-β-Farnesene Synthase(SEQ ID NO:2). E. coil XL1-Blue harboring p43 (including the cDNA insertsequence set forth in SEQ ID NO:1), or empty pBluescript plasmid as acontrol, were grown overnight at 37° C. in LB medium containing 100 μgampicillin/ml. A 50 μl aliquot of the overnight culture was used toinoculate 5 ml of fresh LB medium, and the culture was grown at 37° C.with vigorous agitation to A₆₀₀ 0.5 before induction with 1 mM IPTG.After an additional two hours of growth, the suspension was centrifuged(1000×g, 15 min, 4° C.), the media removed, and the pelleted cellsresuspended in 1 ml of cold assay buffer containing 1 mM EDTA. The cellswere disrupted by sonication with a microprobe as previously described,except that only two 20-second bursts were employed. The chilledsonicate was cleared by centrifugation and the supernatant was assayedfor sesquiterpene synthase activity as before, or for monoterpenesynthase activity (with 4.5 μM [1-³H]GDP) or diterpene synthase activity(with 10 μM [1-³H]GGDP). In all cases, the pentane-soluble reactionproducts were purified by MgSO₄-silica gel chromatography, as above, toprepare the olefin fraction for further analysis.

A cell-free extract of E. coli XL-1 Blue cells harboring the plasmid p43(including the cDNA insert sequence set forth in SEQ ID NO:1) wasprepared and shown to be capable of catalyzing the divalent metalion-dependent conversion of [1-³H]FDP to labeled sesquiterpene olefins.Radio-GC analysis (data not shown) and GC-MS analysis (FIG. 3) of thissesquiterpene olefin fraction demonstrated that the major biosyntheticproduct (85%) was (E)-β-farnesene by matching of both retention time andmass spectrum to those of the authentic standard obtained from severalnatural sources. Lesser amounts of (Z)-μ-farnesene (8%) and δ-cadinene(5%), as well as three other minor products (less than 1% each; allseemingly of the cadinene-type based on MS), were also produced. Controlreactions, employing extracts of XL1-Blue cells transformed withpBluescript lacking the cDNA insert having the sequence set forth in SEQID NO:1, evidenced no detectable production of sesquiterpene olefinsfrom [1-³H]FDP, thereby demonstrating that a cDNA clone encoding(E)-β-farnesene synthase had been acquired.

Multiple product formation is a common feature of the terpenoidsynthases, and may be a consequence of the electrophilic reactionmechanism catalyzed by these enzymes in which highly reactivecarbocationic intermediates are generated (Cane, D. E. (1990) Chem. Rev.90, 1089-1103; Croteau, R. (1987) Chem. Rev. 87, 929-954).(E)-β-farnesene is one of the simplest sesquiterpene olefins that can bederived from FDP, in a reaction involving divalent metal ion-assistedionization of the diphosphate ester and deprotonation from the C-3methyl of the resulting carbocation (FIG. 4). The formation ofδ-cadinene (FIG. 4) involves a considerably more extended reactionsequence, in which a preliminary isomerization step (to nerolidyldiphosphate) is required to permit the ionization-dependent cyclizationto the macrocycle, followed by 1,3-hydride shift, closure of the secondring, and deprotonation to the bicyclic product. The small amount ofδ-cadinene produced by the recombinant synthase (SEQ ID NO:2) from FDPis interesting in light of the abundance of this bicyclic olefin in thesesquiterpene fraction of peppermint oil and the efficient production ofthis olefin in oil gland extracts; these observations suggest that anadditional and distinct δ-cadinene synthase must operate in peppermint.

The recombinant (E)-β-farnesene synthase (SEQ ID NO:2) was inactive withthe C₂₀ substrate analog [1-³H]GGDP, but was able to catalyze thedivalent cation-dependent conversion of the C₁₀ analog [1-³H]GDP tomonoterpene olefins. Although the rate of conversion of GDP to theseproducts was less than 3% of the rate of conversion of FDP tosesquiterpene olefins at saturation, a more diverse spectrum of productswas formed (see FIG. 5 for structures). The cyclic monoterpenes limonene(48%) and terpinolene (15%), and the acyclic monoterpene analog ofβ-farnesene, myrcene (15%), were the most abundant products asdetermined by both radio-GC and GC-MS analysis (data not shown). Lesseramounts of γ-terpinene (7%), (Z)-ocimene (6%), (E)-ocimene (7%), andsabinene (3%) were also observed as products. Control reactions,employing extracts of XL1-Blue cells transformed with pBluescriptlacking the insert, evidenced no detectable production of monoterpeneolefins from [1-³H]GDP, thereby confirming that the monoterpene synthaseactivity expressed from p43 was a function of the (E)-β-farnesenesynthase (SEQ ID NO:2). This is the first report describing theutilization of GDP by a sesquiterpene synthase. Because monoterpenebiosynthesis is localized to plastids, as is diterpene biosynthesis,whereas sesquiterpene biosynthesis occurs in the cytoplasm (Chappell, J.(1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 521-547), theutilization of GDP as a substrate by (E)-β-farnesene synthase isunlikely to be of physiological relevance and may simply reflect thelack of evolutionary pressure to discern the chain length of thisisoprenoid substrate to which the enzyme is not exposed in vivo.

EXAMPLE 3 Sequence Analysis of the p43 cDNA Insert (SEQ ID NO:1)

Complete sequencing of the (E)-β-farnesene synthase cDNA (SEQ ID NO:1)contained in p43 revealed an insert size of 1959 bp encoding an openreading frame of 550 amino acids with a deduced molecular weight of63,829. A putative starting methionine codon was identified which wasout of frame with the vector β-galactosidase starting methionine;however, a fortuitous stop codon in the 5′-untranslated region, 46 bpupstream of the synthase translation start site and in frame with theβ-galactosidase fusion sequence, allowed polycistronic translation ofthe cDNA free of vector-derived sequence. The deduced amino acidsequence of the (E)-βfarnesene synthase (SEQ ID NO:2) lacks a plastidialtargeting peptide (Keegstra, K., Olsen, J J. and Theg, S. M. (1989) Ann.Rev. Plant Physiol. Plant Mol. Biol. 40, 471-501), typical ofmonoterpene and diterpene synthases (Colby, S. M., Alonso, W. R.,Katahira, E. J., McGarvey, D. J. and Croteau, R. (1993) J. Biol. Chem.268, 23016-23024; Stofer Vogel, B., Wildung, M. R., Vogel, G. andCroteau, R. (1996) J. Biol. Chem. 271, 23262-23268; Wildung, M. R. andCroteau, R. (1996) J. Biol. Chem. 271, 9201-9204), but consistent withall known plant-derived sesquiterpene synthases (Fachinni, P. J. andChappell, J. (1992) Proc. Natl. Acad. Sci. USA 89, 11088-11092; Back, K.and Chappell, J. (1995) J. Biol. Chem. 270, 7375-7381; Chen, X. Y.,Chen, Y., Heinstein, P. and Davisson, V. J. (1996) Arch. Biochem.Biophys. 324, 255-266) which are directed to the cytoplasm. Like allother known terpenoid synthases, (E)-β-farnesene synthase (SEQ ID NO:2)is rich in tryptophan (1.8%) and arginine (5.5%) residues, and bears aDDXXD motif (residues 301-305)(SEQ ID NO:3) which is believed tocoordinate the divalent metal ion chelated to the substrate diphosphategroup (Marrero, O. F., Poulter, C. D. and Edwards, P. A. (1992) J. Biol.Chem. 267, 21873-21878); the enzyme (SEQ ID NO:2) has a deducedisoelectric point at pH 5.16.

The deduced amino acid sequence of the farnesene synthase (SEQ ID NO:2)is most similar to that of the sesquiterpene cyclase epi-aristolochenesynthase from tobacco (Fachinni, P. J. and Chappell, J. (1992) Proc.Natl. Acad. Sci. USA 89, 11088-11092) in exhibiting 62% similarity (S)and 49% identity (I). This peppermint synthase (SEQ ID NO:2) alsoclosely resembles the three other known angiosperm sesquiterpenecyclases (vetispiradiene synthase from Hyoscyamus muticus (Back, K. andChappell, J. (1995) J. Biol. Chem. 270, 7375-7381) at 63% S and 40% I,δ-cadinene synthase from cotton (Chen, X. Y., Chen, Y., Heinstein, P.and Davisson, V. J. (1996) Arch. Biochem. Biophys. 324, 255-266) at 60%S and 37% I, and germacrene C synthase from tomato at 57% S and 34% I(unpublished), and also the diterpene cyclase, casbene synthase (Mau, C.J. D. and West. C. A. (1994) Proc. Natl. Acad. Sci. USA 91, 8497-8501),from castor bean (at 61% S and 35% I). Since (E)-β-farnesene synthase(SEQ ID NO:2) produces a small amount of δ-cadinene, but cannot be themajor source of δ-cadinene in peppermint, it is tempting to speculatethat the farnesene synthase (SEQ ID NO:2) represents either aprogenitor, or an altered form of cadinene synthase in which the abilityto catalyze the more complex bicyclization reaction has been lost.

Surprisingly, (E)-β-farnesene synthase (SEQ ID NO:2) is no more closelyrelated to monoterpene synthases from the Lamiaceae (limonene synthasefrom spearmint (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey,D. J. and Croteau, R. (1993) J. Biol. Chem. 268, 23016-23024) with 51% Sand 30% I; sabinene synthase and 1,8-cineole synthase from culinary sagewith 50% S and 29% I each) than to the various terpenoid synthases fromthe gymnosperm Abies grandis (monoterpene synthases with 49% S and 28% I(Bohlmann, J., Steele, C. L. and Croteau, R. (1997) J. Biol. Chem. 272,21784-21792); sesquiterpene synthases with 53% S and 29% I; diterpenesynthases with 51% S and 28% I (Stofer Vogel, B., Wildung, M. R., Vogel,G. and Croteau, R. (1996) J. Biol. Chem. 271, 23262-23268). Even aphylogenetically distant diterpene cyclase from Taxus brevifolia,taxadiene synthase (Wildung, M. R. and Croteau, R. (1996) J. Biol Chem.271, 9201-9204), resembles (E)-β-farnesene synthase (SEQ ID NO:2) at theamino acid level (50% S and 24% I) as closely as do the monoterpenesynthases of the mint family. These sequence-based relationships mayreflect a bifurcation in the evolution of the monoterpene synthases fromthe higher terpenoid synthases that is as ancient as the separationbetween the angiosperms and gymnosperms.

EXAMPLE 4 Characterization of (E)-β-Farnesene Synthase (SEQ ID NO:2)

For determination of the pH optimum of (E)-β-farnesene synthase (SEQ IDNO:2), the preparation was adjusted with 50 mM Mopso (to a pH of 6.5,6.75, 7.0, 7.25, 7.5, 8.0, or 8.5) before the assay. Kinetic constantsfor FDP, GDP, Mg⁺⁺ and Mn⁺⁺ were determined using a preparation of(E)-β-farnesene synthase (SEQ ID NO:2) that was partially purified byanion-exchange chromatography (on a Mono-Q column (Pharmacia)equilibrated with assay buffer and eluted with a linear KCl gradient (0to 500 mM) in assay buffer). The 210-230 mM fraction containing the(E)-β-farnesene synthase (SEQ ID NO:2) was used for kinetic evaluationof FDP and GDP as substrates (concentration range 0.31 to 20 μM, withsaturating Mg⁺⁺). Due to the tenacious binding of divalent cations bythe synthase, the partially purified enzyme (prepared in the presence of10 mM EDTA) was dialyzed overnight against assay buffer containing 50 mMEDTA. The dialysate was buffer-exchanged by ultrafiltration (AmiconCentriprep 30, 450 fold dilution), then desalted (Bio-Rad Econo-Pak 10DG) into assay buffer. Kinetic constants for Mg⁺⁺ and Mn⁺⁺ (assay range1 μM to 2 mM of the chloride salts) were then determined at 7.3 μM[1-³H]FDP. Triplicate assays were conducted and control incubations(without enzyme) were included in all cases. A double reciprocal plot(Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc. 56, 658-666) wasgenerated for each averaged data set, and the equation of the best-fitline determined (Kaleidagraph ver. 3.08, Synergy Software).

The recombinant, partially purified (E)-β-farnesene synthase (SEQ IDNO:2) exhibited a broad pH optimum in the 6.75 to 7.25 range in Mopsobuffer. This observation is in agreement with the studies of Salin etal. (Satin, F., Pauly, G., Charon, J. and Gleizes, M. (1995) J. PlantPhys. 146, 203-209) in which the purified (E)-β-farnesene synthase frommaritime pine was shown to possess a pH optimum in the 7.0 to 7.3 range.The K_(m) value for FDP with the recombinant synthase (SEQ ID NO:2) wascalculated to be 0.6 μM, a value typical of other sesquiterpenesynthases of plant origin (Cane, D. E. (1990) Chem. Rev. 90, 1089-1103)but lower than the value of 5 μM determined for the enzyme from maritimepine (Salin, F., Pauly, G., Charon, J. and Gleizes, M. (1995) J. PlantPhys. 146, 203-209). Substrate concentrations in excess of 10 μM FDPevidenced slight inhibition of activity with the recombinant enzyme (SEQID NO:2). Although the relative velocity at saturating levels of GDP wasonly 3% of the velocity with FDP for the recombinant synthase (SEQ IDNO:2), the calculated Km value for GDP (1.5 μM) was only three-foldhigher than that for FDP, suggesting that the binding of the C₁₀ analogwas reasonably efficient.

A K_(m) value of 150 μM was determined for Mg⁺⁺ (V_(rel)=100), and aK_(m) value of 7.0 μM was determined for Mn⁺⁺ (V_(rel)=80). Noinhibition of activity was observed at Mg⁺⁺ concentrations up to 10 mM;however, concentrations of Mn⁺⁺ exceeding 20 μM resulted in a sharpdecline in reaction velocity to a plateau (V_(rel)=20) in the 0.25 to 2mM range. Since the product distribution of the recombinant(E)-βfarnesene synthase (SEQ ID NO:2) had been initially determined inthe presence of excess Mg⁺⁺, the conversion of [1-³H]FDP wasre-evaluated in the presence of Mn⁺⁺ alone at apparent saturation (20μM). The olefin products were again analyzed by GC-MS and found in thiscase to consist of 98% (E)-β-farnesene and 2% (Z)-β-farnesene. Noδ-cadinene, or other sesquiterpenes, were synthesized in this instance,indicating that a structural alteration in the binding of Mn⁺⁺ to thesubstrate and/or enzyme (relative to Mg⁺⁺) improves the fidelity of thereaction.

In operational characteristics (pH optimum, kinetic constants) andphysical features (size, pI), the recombinant (E)-β-farnesene synthase(SEQ ID NO:2) is a typical sesquiterpene synthase (Cane, D. E. (1990)Chem. Rev. 90, 1089-1103; Fachinni, P. J. and Chappell, J. (1992) Proc.Natl. Acad. Sci. USA 89. 11088-11092; Back, K. and Chappell, J. (1995)J. Biol. Chem 270, 7375-7381; Clien, X. Y., Chen, Y., Heinstein, P. andDavisson, V. J. (1996) Arch. Biochem. Biophys. 324, 255-266), suggestingthat the enzyme should be highly functional in planta. Given that thissynthase (SEQ ID NO:2) will be targeted by default to the cytoplasm(Chappell, J. (1995) Annu. Rev. Plant Physioll. Plant Mol. Biol. 46,521-547; Keegstra, K., Olsen, J. J. and Theg, S. M. (1989) Ann. Rev.Plant Physiol. Plant Mol. Biol. 40, 471-501), where the substrate arisesfrom the mevalonate pathway, it should be possible to engineer virtuallyany plant for the production of (E)-β-farnesene in order to exploit thekairomonal and pheromonal properties of this natural product.

EXAMPLE 5 Properties of (E)-β-Farnesene Synthase Proteins of the PresentInvention

The (E)-β-farnesene synthase proteins of the present invention allrequire a divalent metal ion as a cofactor. Most (E)-β-farnesenesynthase proteins of the present invention utilize either Mg⁺⁺ or Mn⁺⁺as a cofactor. Nonetheless, (E)-β-farnesene synthase proteins of thepresent invention are inhibited at concentrations of Mn⁺⁺ in excess ofabout 5 mM.

(E)-β-farnesene synthase proteins of the present invention have a pHoptimum in the range of from about pH 5.5 to about pH 8.5, and a pI inthe range of from about pH 4.5 to about pH 6.0. The Km(FPP) of(E)-β-farnesene synthase proteins of the present invention is less thanabout 10 μM, while the Kcat(FPP) of (E)-β-farnesene synthase proteins ofthe present invention is less than about 5/sec. The (E)-β-farnesenesynthase proteins of the present invention exist as either monomers orhomodimers, with the monomer having a molecular weight of from about 55kD (kiloDaltons) to about 65 kD.

EXAMPLE 6 Hybridization of Peppermint (E)-D3-Farnesene Synthase cDNA(SEQ ID NO:1) to other Nucleic Acid Sequences of the Present Invention

The nucleic acid molecules of the present invention are capable ofhybridizing to the nucleic acid sequence set forth in SEQ ID NO:1, or tothe complementary sequence of the nucleic acid sequence set forth in SEQID NO:1, under the following stringent hybridization conditions:incubation in 5×SSC at 65° C. for 16 hours, followed by washing underthe following conditions: two washes in 2×SSC at 18° C. to 25° C. fortwenty minutes per wash; preferably, two washes in 2×SSC at 18° C. to25° C. for twenty minutes per wash, followed by one wash in 0.5×SSC at55° C. for thirty minutes; most preferably, two washes in 2×SSC at 18°C. to 25° C. for fifteen minutes per wash, followed by two washes in0.2×SSC at 65° C. for twenty minutes per wash.

The ability of the nucleic acid molecules of the present invention tohybridize to the nucleic acid sequence set forth in SEQ ID NO:1, or tothe complementary sequence of the nucleic acid sequence set forth in SEQID NO:1, can be determined utilizing the technique of hybridizingradiolabelled nucleic acid probes to nucleic acids immobilized onnitrocellulose filters or nylon membranes as set forth, for example, atpages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2ndedition), J. Sambrook, E. F. Fritsch and T. Maniatis eds, the citedpages of which are incorporated herein by reference.

In addition to the nucleic acid sequence set forth in SEQ ID NO:1,examples of representative nucleic acid sequences of the presentinvention that encode a peppermint (E)-β-farnesene synthase protein andwhich hybridize to the complementary sequence of the nucleic acidsequence disclosed in SEQ ID NO:1 are set forth in SEQ ID NO:4; SEQ IDNO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ IDNO:16 and SEQ ID NO:18. With the exception of the nucleic acid sequenceset forth in SEQ ID NO:1, the foregoing representative nucleic acidsequences of the present invention were generated using a computer. Byutilizing the degeneracy of the genetic code, each of the foregoing,representative nucleic acid sequences has a different sequence, but eachencodes the protein set forth in SEQ ID NO:2. Thus, the identical(E)-β-farnesene synthase protein sequence is set forth in SEQ ID NO:2,SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQID NO:15; SEQ ID NO:17 and SEQ ID NO:19.

In addition to the protein sequence set forth in SEQ ID NO:2 examples ofrepresentative (E)-β-farnesene synthase proteins of the presentinvention are set forth in SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQID NO:23; SEQ ID NO:24, SEQ ID NO:25; SEQ ID NO:26, SEQ ID NO:27 and SEQID NO:28. With the exception of the amino acid sequence set forth in SEQID NO:2, the foregoing representative amino acid sequences of thepresent invention were generated using a computer by making conservativeamino acid substitutions.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A recombinant (E)-β-farnesene synthase protein encoded by an isolatednucleic acid molecule that hybridizes to the nucleic acid sequence ofSEQ ID NO:1, or to the complement thereof, under conditions of 0.5×SSCat 55° C.
 2. A recombinant (E)-β-farnesene synthase protein of claim 1wherein said recombinant (E)-β-farnesene synthase protein has anisoelectric point in the range of from about pH 4.5 to about pH 6.0. 3.A recombinant (E)-β-farnesene synthase protein of claim 1 that comprisesthe amino acid sequence DDXXD.
 4. A recombinant (E)-β-farnesene synthaseprotein of claim 1 comprising the amino acid sequence set forth in SEQID NO:2.
 5. An (E)-β-farnesene synthase protein of claim 1 said proteinconsisting of the amino acid sequence set forth in SEQ ID NO:2.